【作者】 周岩；

【导师】 范洪学；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2010， 博士

【摘要】 扩张型心肌病(dilated cardiomyopathy,DCM)是一种临床常见的心肌病,该病预后差,5-10年生存率较低(30%-40%)。DCM的治疗目前尚无特效药物及方法。干细胞移植是治疗DCM的一种新方法,已经成为国内外研究的热点。骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)是存在于骨髓中的非造血实质的基质干细胞,具有来源广泛、取材方便、在体外容易扩增、具有较强的自我复制和多向分化能力、免疫源性弱、可以自体移植、不存在伦理方面的问题、可以进行基因修饰、能分化为心肌细胞和内皮细胞等优势,BMSCs已经成为心血管疾病细胞移植治疗的理想种子细胞。本研究旨在探讨在体外不同诱导条件下BMSCs分化为心肌细胞的作用;建立稳定可靠的DCM动物模型,以供基础实验研究;探讨BMSCs移植治疗DCM模型大鼠的作用及机制。本研究采用密度梯度离心法结合贴壁培养法分离培养大鼠BMSCs,在体外分别用5-氮胞苷(5-azacytidine, 5–aza)、DCM模型大鼠血清+5–aza、DCM模型大鼠心肌细胞裂解液等作为条件培养液培养,观察BMSCs形态变化、用免疫组化和RT-PCR法检测心肌肌钙蛋白T(cardiac troponin T;CTnT)的表达情况。选用雄性Wistar大鼠,接受腹腔注射阿霉素的方法建立DCM模型。然后用5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine ,Brdu)标记BMSCs、DCM模型大鼠血清+5–aza培养诱导的细胞进行细胞移植,采用单次静脉注射和重复静脉注射的方法,观察各组大鼠的状态及死亡率、超声心动图、脑利钠肽(brain natriuretic peptide,BNP)水平、病理改变、Masson染色结果、用激光共聚焦显微镜追踪移植细胞在心脏中的分布情况和分化情况。用免疫组化和RT-PCR法检测血管内皮生长因子(vascular endothelial growth factor,VEGF)表达情况。本研究得出的结论是:密度梯度离心法结合贴壁培养法可以分离培养到大量、高活性、纯化的BMSCs;在体外用5–aza、DCM大鼠模型的心肌细胞裂解液、DCM大鼠模型血清+5–aza三种方法均可诱导BMSCs向心肌细胞分化,其中DCM模型大鼠血清+5–aza(5μmol/L)诱导组细胞生长状态、向心肌细胞分化的结果最佳;用阿霉素2.5mg/(kg·week),连续6周腹腔注射给药,建立DCM大鼠模型的方法成功率高,造模周期较短,结果可靠,可以做为典型、稳定的供基础研究的动物模型;经静脉移植是一种有效的移植途径,重复静脉治疗提高了移植细胞的数量、延长了补充供体细胞的时间,治疗效果优于单次静脉移植治疗;直接用BMSCs移植治疗和用先经体外诱导分化后的BMSCs移植治疗均能改善心功能,两者无显著差异;BMSCs在体内可以分化为心肌细胞,并表达心肌特异性cTnT;BMSCs移植治疗可以提高DCM大鼠的存活率、改善心功能、抑制心肌纤维化、增加VEGF的表达,改善心室重构。更多还原

【Abstract】 Dilated cardiomyopathy (DCM) is a clinical common cardiac disease. Its etiology may be related to infection, autoimmune, metabolic abnormalities and family history. It is mainly characterized by dilatation and systolic dysfunction of left or bilateral ventricular. It is histologically marked by myocardial cell hypertrophy, degeneration, mitochondrial function defect, and different levels of fibrosis. The prognosis is poor, and 5-10-year survival rate is low (30% to 40%). Up to now, there is no specific drug treatment and methods. The current therapy is to control heart failure, lessen symptom and prevent ventricular remodeling, including the use of medicines, such as angiotensin converting enzyme inhibitor (ACEI), diuretics, digoxin,βblockers, calcium antagonists and other drugs. As the poor prognosis of the DCM patients, heart transplantation is the most effective treatment of patients with DCM in advanced stage, but many problems have been encountered in clinical practice, such as donor source, immunological rejection and costs. So cardiologists have been looking for a new treatment method. Stem cell transplantation as a new treatment of DCM has became a hot research area in recent years. Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic stem cells in the bone marrow with a variety of sources, drawing convenience, easy expansion in vitro, pluripotency, and possess a weak immune rejection, and can be autologous transplantation with no ethical problems, and can be genetically modified, and can differentiate into myocardial cells and endothelial cells and other advantages. So BMSCs have become ideal seed cells for cell transplantation to treat cardiovascular disease. The treatment of acute myocardial infarction using stem cell transplantation has unparalleled advantages compared with conventional treatment, while the researches about the therapy of non-ischemic cardiovascular diseases with the BMSCs are less. Meanwhile many problems still remains, such as the choice of seed cells, the quantity of transplanted cells, the path of transplantation, treatment time, the duration of BMSCs proliferation and survival in the recipient, and how to improve the differentiation capabilities of BMSCs, when to follow-up after treatment, security in clinical application, which are need to be solved.Objective1. To explore the effect of differentiation of BMSCs into myocardial cells in different inducing conditions in vitro.2. To establish a stable and reliable animal model with DCM for basic experimental research.3. To explore the effect of BMSCs on the therapy of DCM with different transplantation methods and its mechanismMethods1. BMSCs were isolated with the density gradient centrifugation combined with adherent method and cultured. The growth curve, cell cycle, phenotype of BMSCs were determined. BMSCs were induced to differentiate into osteoblasts and adipocytes. In vitro BMSCs were cultured in the medium supplemented with 5–azacytidine(5–aza) or serum of rat with DCM combined with 5–aza or cadiocyte lysate of rat with DCM. Then the morphological changes were observed and the expression of cardiac troponin was detected using the methods of immunocytochemistry and RT-PCR.2. Rats DCM models were established by the following method. Male Wistar rats were administered doxorubicin 2.5 mg / (kg ? week) by intraperitoneal injection for 6 weeks. Then rats were observed for 2 weeks after the withdrawal. While rats in the control group were given the same volume normal saline with the same exposure route. The body weight changes and mortality were recorded, and echocardiography was performed. In addition to, plasma brain natriuretic peptide (BNP) level was detected, left ventricular internal diameter was measured, and pathological observation, electron microscopy, Masson staining of myocardium were performed.3. BMSCs labeled with Brdu and differentiated cells derived from the BMSCs in the medium supplemented with the serum of rat with DCM combined 5–aza were transplanted using a single intravenous injection and repeated intravenous injection method. The rats were randomly divided into normal control group (12 rats), model control group (17 rats), treatment group 1 (12 rats) which represents the single intravenous injection with BMSCs , treatment group 2 (12 rats) which represents the repeat intravenous injection with BMSCs, treatment group 3 (12 rats) which represents the single intravenous injection with differentiated cells cultured with the serum of rat with DCM combined 5–aza, treatment group 4 (12 rats) which represents the repeat intravenous injection with the same cells as in the treatment group 3. The state and mortality of the rats were observed, echocardiography, BNP levels, pathological observation and Masson staining were performed. The distribution and differentiation of transplanted cells in the heart were traced with laser scanning confocal microscope. The expression of VEGF was detected using immunohistochemistry and RT-PCR.Results1. Most of BMSCs isolated with the density gradient centrifugation combined adherent method and cultured adhered to the wall with the colony growth. Passaged cells quickly adhered, proliferated rapidly, and almost no colonies were formed. About 6 - 7d later, the cells crowded the bottom of the flask with the spindle-shaped uniform distribution. After many passages, cells have a more uniform and orderly distribution of fibroblast-like. After passaged for consecutive 9 times, cells had no significant changes in morphology, no signs of aging. The passage cycle is 6-7d, but after the 9 passage, the proliferation of cells was slow and the passage cycle was extended. The Passage1, 3, and 5 cells were high proliferation, the growth curve shape of which were similar. The majority of the cells were relatively quiescent. BMSCs could differentiate into osteoblasts and to fat cells under particular induced conditions. After the phenotypes of BMSCs were determined by flow cytometry, they were CD44-positive andCD34-negative. All the three methods could induce BMSCs to differentiate into cardiocytes-like cells with the expression of troponin, among which the growth state of cells in 5–aza group was poor; the cells in the other two groups grew better. The positive-expression rate of cardiac troponin of differentiated cells in the serum of rat with DCM combined 5–aza group was highest.2.The bodyweight was significantly different between normal group and model group on the 8th weeks. Rat mortality rate in the model group was 16.7%. The echocardiography findings, plasma BNP, left ventricular internal diameter were significantly different between normal group and model group. In the normal group, the results of HE showed that cardiac muscle fibers arranged regularly, no myocardial fiber breakage, uniform and rich cytoplasm, normal cell space and no edema. The results of electron microscopy displayed myocardial fibers arranged regularly, the uniform size of mitochondria, no edema, intact membrane and normal density of crest. The results of myocardial Masson staining showed collagen distributing sparse and complete collagen fibers nets of the neighbor cells and pale staining. In the model group, the results of HE staining showed myocardial fibers disorganized, myocardial fiber breakage, widened cell gap, edema and vacuole degeneration. The results of electron microscopy showed myocardial fiber fracture, swelling mitochondria, vacuole degeneration, and crest depletion. The results of myocardial Masson staining showed increased collagen in myocardium, breakage and derangement of collagen fibers surrounding the myocardial cells.The CVF compared with the normal group had significantly different.3. In model group without cell transplantation, the state of rats were poor, and body weight did not increase, seven rats died, and the mortality rate was 41.2%. While the state of rats in cell transplantation groups were improved, the body weight increased, the mortality of rats in all the cell transplantation groups was lower than that in the model group. The mortality rate of rats in repeat intravenous injection group was lower than that in single intravenous injection group. The cardiac function with echocardiography 2 weeks after cell transplantation had no significantly difference (p> 0.05) between treatment groups and model group. The results of cardiac function with echocardiography and plasma BNP 4 weeks after cell transplantation were: the results of treatment group 1, 2, 3, 4 were significantly different compared with that in the control group (p <0.05); the results in treatment group 2 and 4- were significantly different from that in the model group (p<0.05); there were significant difference between the treatment group 1 and treatment group 2 (p <0.05); there were significant difference between treatment group 3 and treatment group 4 (p <0.05); there was no significant difference between the treatment group 1, 3 and model group (p>0.05); there was no significant difference between treatment group 2 and treatment 4 (p> 0.05). The distribution and cTnT expression of transplant cells in myocardial tissue using the laser confocal microscope showed that transplanted cells labeled with Brdu could be found in the treatment group 1, 2, 3, 4, which distributed disperse, fused with myocardial cell, and expressed cTnT. However, the numbers of the transplanted cells labeled with Brdu in repeated intravenous injection treatment group 2, 4 were more than that in the single intravenous injection treatment group 1, 3, and the expression of cTnT in cells was significantly increased. There was no significant difference between treatment group 1 and treatment group 3 and between treatment group 2 and treatment group 4. The results of HE staining showed that myocardial cells still arranged disordered, myocardial fiber breakage was alleviated in treatment group 1 and treatment group 3 compared with model group. However, some changes still remained, such as widened cell gap, cell edema and vacuole degeneration in treatment group 1 and treatment group 3.The result of HE staining displayed that myocardial cells arrangement were no obvious disorder, no significant cardiac fiber broke, cell gap widened, edema and degeneration occurred, which significantly were alleviated compared with that in the model group in treatment group 2 and treatment group 4. Masson staining results showed there was still more myocardial collagen, and collagen fiber was still broken and disordered in treatment group 1 and treatment group 3. While there was less myocardial collagen in treatment group 2 and treatment group 4 than that in model group. In treatment group 2 and treatment group 4, there was no significant collagen fiber breakage and disorderly arrangement, and expression of VEGF was higher in treatment group 2 and treatment group 4 than that in other groups by immunohistochemistry and RT-PCR.Conclusion1. A number of highly active purified BMSCs were isolated by density gradient centrifugation combined with adherent method. 2. In vitro the three methods can induce BMSCs to differentiate into cardiocyte-like cells, which include 5–aza, the serum of rat with DCM model combined with 5–aza, the myocardium lysate of rat with DCM.3. The induced effects were better in the medium supplemented with the serum of rat with DCM combined with 5–aza, the myocardium lysate of rat with DCM than that with 5–aza. The cells growth state and differentiation into cardiocyte-like cells were the best in the medium supplemented with serum of rat with DCM combined with 5–aza (5μmol / L).4. The method with doxorubicin 2.5mg/(kg ? week) for 6 weeks by intraperitoneal injection to establish rat model with DCM was high success rate, shorter cycle of establishing models and reliable, which can be used as preparing animal models for basic research.5. The vein transplantation was an effective way, repeated intravenous treatment can increase the number of transplanted cells, extend the time of add donor cells, and its effect was better than that in single intravenous treatment.6. There were no significant difference between BMSCs transplantation treatment and transplantation treatment of cardiocyte-like cells differentiated from BMSCs.7.In vivo BMSCs can differentiate into myocardial cells, which expressed cardiac-specific cTnT.8. BMSCs transplantation for treatment of DCM in rats can reduce mortality and improve cardiac function, inhibit myocardial fibrosis, increase expression of VEGF and improve ventricular remodeling.更多还原